1. Field of the Invention
The invention relates generally to a device and method for the printing of high density arrays for use in biological and chemical assays in the form of a device that can be used in sample transfer directly from a standard multiple-well microassay plate to a substrate.
2. Background of the Invention
Biomolecules, such as peptides or oligonucleotides, immobilized on planar substrates are increasingly useful as diagnostic or screening tools. Such xe2x80x9cbioarraysxe2x80x9d include regions of usually different biomolecules arranged in a predetermined configuration on the substrate. These regions (sometimes referenced as xe2x80x9cfeaturesxe2x80x9d or xe2x80x9cspotsxe2x80x9d) are positioned at respective locations (xe2x80x9caddressesxe2x80x9d) on the substrate. The arrays, when exposed to a sample, will exhibit an observed binding pattern. This binding pattern can be detected upon interrogating or imaging the array. Data obtained from the interrogation or imaging process are then analyzed to determine information about the sample. For example, all polynucleotide targets (e.g. DNA) in a sample can be labeled with a suitable label (such as a fluorescent compound) to provide a suitable binding signal during the interrogation or imaging process, and the labeled sample can then be assayed using an oligonucleotide array. Following exposure to the labeled sample, the fluorescence pattern on the array can be accurately observed. Assuming that the different sequence oligonucleotides were correctly deposited in accordance with the predetermined configuration, then the observed binding pattern will be indicative of the presence and/or concentration of one or more polynucleotide components of the sample.
Bioarrays can be fabricated by depositing previously obtained biomolecules onto a substrate surface, or by in situ synthesis of the biomolecule on the substrate surface. Fabrication of bioarrays typically involves deposition of very small droplets of reagent solutions onto the substrate surface. Both manual and automated devices for dispensing very small fluid volumes have been devised, including, for example, micropipettes, pins, capillaries, and inkjet devices. One way of making bioarrays that has been described involves depositing dots of fluid chemicals on a substrate surface using a rod that pokes out of a capillary and touches off a drop of fluid onto the substrate. While the art contains a variety of references related to this technique, each of these is associated with certain disadvantages.
U.S. Pat. No. 2,643,801 to Kollmeyer, xe2x80x9cWatch Oilerxe2x80x9d (1953) describes a rigid capillary tube through which a rigid wire extends and an on-axis reservoir surrounding the wire for depositing a drop of oil on a jeweled bearing in a watch. The device touches off a drop of oil from the oiler to the bearing without direct contact between the oiler and the bearing.
U.S. Pat. No. 3,334,354 to Mutschler, xe2x80x9cDotting Ink Recorderxe2x80x9d (1967) describes a dot printer with a rigid capillary tube through which a rigid pin extends, and on-axis reservoir surrounding the pin and fed by an off-axis non-capillary fill port. The invention avoids splashing during printing by touching only the fluid on the tip of the pin, rather than the pin itself, to the paper substrate on which printing occurs.
U.S. Pat. No. 4,194,846 to Zerillo, xe2x80x9cDot Matrix Printing Device Employing a Novel Image Transfer Technique to Print on Single or Multiple Ply Print Receiving Materialsxe2x80x9d (1980), assigned to Centronics Data Computer Corp., describes a dot printer with a rigid capillary tube through which a flexible pin extends, and an off-axis ink reservoir feeding the same color ink to multiple flexible pins extending through a rigid matrix.
International patent application WO 00/54883, xe2x80x9cApparatus and Method for Spotting a Substrate,xe2x80x9d applied for by Perkin-Elmer Corp., describes a dot printer with a tube through which a flexible pin extends and an on-axis reservoir surrounding the pin.
European patent application EP 1002570(A1) and international patent application WO 00/30754, xe2x80x9cCapillary Transfer Device for High Density Arrays,xe2x80x9d applied for by Corning Inc., describe a dot printer with a rigid capillary tube through which a flexible pin extends, and an on-axis reservoir surrounding the pin. The pin must be removed from the capillary tube in order to fill the reservoir. A plurality of tubes and reservoirs are formed in a rigid matrix. This application also describes touching the flat-faced pins to the substrate or expelling fluid across open space in order to print, which risks splashing fluid.
With the exception of the noted EP ""570 patent, the above-cited references teach pin-in-capillary printers that can deposit drops without splashing, avoiding resultant cross-contamination between drops. But none of the above printers are easy to load with the expensive fluids that the bioscience industry prepares in standard plastic microassay plates containing, e.g., 96, 384, or 1536 separate wells, with each well having a fluid volume on the order of microliters.
U.S. Pat. No. 6,083,763 to Balch, assigned to Genometrix, and associated international patent application WO98/29736(A1), describe a capillary printer comprising a flexible capillary extending from a point near a substrate to be printed to a reservoir which is on-axis with the capillary. Multiple capillaries are bundled together and extend through a rigid guide matrix through which the capillaries slide. Advantageously, multiple capillaries can be fed from multiple reservoirs comprising a standard multiple well microassay plate. However, the capillaries have a large diameter in comparison to the pin of the above-described pin-in-capillary printers, leading to lower density of dots and larger dot size. Also, the required free length of capillary for needed flexing (that is, elastic buckling) during printing to accommodate surface irregularity of the substrate is larger than the required free length of thin pins for needed flexing in comparable pin-in-capillary schemes, leading to potentially greater size and mass of the reciprocating portion of the apparatus than in comparable pin-in-capillary schemes. Further, capillaries without internal sliding pins can be more difficult to clean in the event of clogs than pin-in-capillary devices, where the pin can act like a pipe cleaner to dislodge deposits within the capillary.
Thus there still exists a need for a multiple-fluid pin-in-capillary printer where multiple incoming fluids can be conveyed directly from multiple reservoirs (such as in a standard multiple-well microassay plate) to the closely-spaced tips of the pins near the substrate, in a manner leading to low overall size and mass of the reciprocating portion of the apparatus.
The invention addresses the aforementioned deficiencies in the art, and provides novel methods for printing high density biological or chemical arrays on substrates using a fluid droplet delivery apparatus. The method and apparatus of the invention use one or more flexible pins threaded through flexible capillaries, wherein each capillary has a uniform cross sectional area along its length. Each capillary, with the pin threaded through it, is narrow enough to retain fluid by capillary action without drooling. Fluid is expelled from a capillary by extending the tip of the pin from an end of the capillary, whereupon the wetted tip, or the fluid wetting the pin tip, can then touch a substrate to transfer a droplet of fluid to the substrate. Greater fluid volumes may be delivered by retracting the pin further within the capillary and quickly translocating the pin so the pin will, piston-like, pump fluid out of the capillary. Advantageously, each pin may be extended individually, or pins may be extended in associated groups, and the substrate can be moved beneath the capillary ends, so that the density of deposited droplets on the substrate can be greater than the packing density of the pin tips near the substrate.
Far from the substrate, the capillaries can fan out to a density low enough to permit fluid loading through an attached fluid flow manifold. The fluid flow manifold has fluid source channels in fluid communication with the capillary lumens via off-axis ports. The fluid source channels are also in fluid communication with one or more reservoirs, such as wells in a microtiter plate. Each pin extends from its tip at the downstream end of the capillary near the substrate and upstream through the capillary lumen, to operatively connect with a pin driver such as a solenoid driver. Advantageously, an air gap region may be present at the upstream end of the capillary to prevent the fluid in the capillary from welling upward to the top surface of the manifold. The pin may also have an elastic buckling region that accommodates for substrate irregularities. The pins need not be removed from the capillaries during fluid loading, and the required priming volume is low, allowing reduced setup costs, cost-competitive small production runs, and minimal use of expensive chemicals. The invention provides for off-axis loading of a pin-in-capillary printer where each capillary/pin combination may be in communication with a separate reservoir. Advantageously, because the separate flexible capillaries do not form a rigid matrix, the spatial ordering of the outlet ends of the capillaries can be different than the spatial ordering of the input ends of the capillaries, providing additional freedom in spatial reformatting, e.g. between the source microassay plates and the resulting microarrays.
Additional objects, advantages, and novel features of this invention shall be set forth in part in the descriptions and examples that follow and in part will become apparent to those skilled in the art upon examination of the following specifications or may be learned by the practice of the invention. The objects and advantages of the invention may be realized and attained by means of the instruments, combinations, compositions and methods particularly pointed out in the appended claims.